CN110951821B - Rhodobacter sphaeroides mutant strain for producing coenzyme Q10 and method for producing coenzyme Q10 through fermentation - Google Patents
Rhodobacter sphaeroides mutant strain for producing coenzyme Q10 and method for producing coenzyme Q10 through fermentation Download PDFInfo
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Abstract
The invention discloses a Rhodobacter sphaeroides mutant strain for producing coenzyme Q10 and a method for producing coenzyme Q10 by fermentation, wherein the mutant strain is named as Rhodobacter sphaeroides YLL-13-T with the preservation number of CGMCC No.18983, the mutant strain has higher growth rate and coenzyme Q10 yield, and under the optimized culture medium and culture conditions, the biomass of the mutant strain, the coenzyme Q10 yield and the coenzyme Q10 yield are respectively improved by 12.3%, 42.5% and 23.6% compared with the original strain, so that the Rhodobacter sphaeroides mutant strain has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a rhodobacter sphaeroides mutant strain for producing coenzyme Q10 and a method for producing coenzyme Q10 through fermentation.
Background
Coenzyme Q10(Coenzyme Q10, CoQ10), the chemical name of which is 2, 3-dimethoxy-5-methyl-6-decaisopentenylbenzoquinone, is widely distributed on biological membranes in various organisms and is an important electron acceptor in a cell respiratory chain. Coenzyme Q10 is a natural antioxidant and a cell metabolism activator, can prevent and treat various diseases, and has wide application in the aspects of resisting fatigue, treating heart failure, delaying skin aging and the like. Therefore, the realization of the efficient synthesis of the coenzyme Q10 has important economic value.
The production method of coenzyme Q10 mainly comprises four methods, respectively: animal and plant extraction method, chemical synthesis method, plant cell culture method and microorganism fermentation method. Compared with the first three methods, the microbial fermentation method has the advantages of rich raw materials, low cost, relatively simple separation process, good product activity, capability of scale amplification and the like, and becomes the coenzyme Q10 production method with the most development potential. The main factor for restricting the production of coenzyme Q10 by a microbial fermentation method at present is that the yield of the product is not high due to the physiological and genetic characteristics of strains and complicated fermentation regulation and control. The acquisition of a strain with high basic quantity of coenzyme Q10 and the optimization of a fermentation process are one of the most effective and direct means for reducing the industrial production cost of the coenzyme Q10.
At present, a plurality of strains producing coenzyme Q10 are reported and distributed in the species of aspergillus fumigatus, candida, pseudomonas, agrobacterium, paracoccus denitrificans, rhodobacter capsulatus, rhodobacter sphaeroides and the like. Among them, rhodobacter sphaeroides is the most widely noticed. The process of rhodobacter sphaeroides synthesizing coenzyme Q10 is mainly divided into MEP pathway, shikimic acid pathway and coenzyme Q10 pathway. Therefore, strengthening the above way, weakening branch flux and removing product inhibition has important value for improving the fermentation level of rhodobacter sphaeroides coenzyme Q10. Vitamin K3 and p-hydroxybenzoic acid are coenzyme Q10 analogues and precursors respectively, therefore, the invention uses nitrosoguanidine as a chemical mutagen, and uses vitamin K3 and p-hydroxybenzoic acid as screening markers to screen mutant strains with low carotenoid synthesis, high growth rate and high coenzyme Q10 accumulation, and optimizes the culture medium and culture conditions.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide a rhodobacter sphaeroides mutant strain with high coenzyme Q10 yield and a screening and obtaining method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for screening rhodobacter sphaeroides with high coenzyme Q10 yield, which comprises the following steps:
1) inoculating an original strain into a seed culture medium, culturing for 24h at 32 ℃ under the condition of 200r/min, taking a bacterial suspension, centrifuging to obtain thalli, performing centrifugal washing twice, then suspending in physiological saline containing a chemical mutagen, and standing at room temperature;
2) centrifuging to obtain thallus, re-suspending in normal saline, spreading onto solid culture medium plate containing coenzyme Q10 analogue and/or coenzyme Q10 precursor analogue, and standing at 32 deg.C for culture;
3) selecting a mutant strain with large bacterial colony, relatively light color and relatively low bacterial colony viscosity on a flat plate, culturing for 120h at 32 ℃ under 200r/min in a seed culture medium, and measuring the content of the cell coenzyme Q10 to obtain the mutant strain with high coenzyme Q10 accumulation.
Further, the original strain is rhodobacter sphaeroides YLL-13 separated from sludge of a sewage treatment plant.
Further, the seed medium comprises: 3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, NaCl2.0g/L KH2PO41.3g/L，MgSO4125mg/L，FeSO4100mg/L，MnSO41.0mg/L, biotin 15 mug/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2; the solid medium comprises: 3g/L glucose, 8g/L yeast powder, 2g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L, biotin 15 mu g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, agar powder 15g/L, and pH 7.2.
Further, the chemical mutagen in 1) is nitrosoguanidine, preferably, the content of the nitrosoguanidine is 0.6 mg/mL.
Further, 1) standing at room temperature for 45 min.
Further, the coenzyme Q10 structural analogue in the step 2) is vitamin K3, and the coenzyme Q10 precursor analogue is p-hydroxybenzoic acid.
Further, the content of the vitamin K3 is 12mg/L, and the content of p-hydroxybenzoic acid is 0.6 g/L.
Further, 2) standing at 32 ℃ for 4-5 days.
Furthermore, the method also comprises the steps of subculturing the screened rhodobacter sphaeroides with high coenzyme Q10 yield, screening mutant strains with good genetic stability and preserving the mutant strains.
In a second aspect, the invention provides a Rhodobacter sphaeroides mutant strain obtained by the method of the first aspect, which is named Rhodobacter sphaeroides (Rhodobacter sphaeroides) YLL-13-T, and the Rhodobacter sphaeroides mutant strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 18983.
Furthermore, the 16S rDNA gene sequence of the mutant strain is shown in SEQ ID NO. 1.
Further, the YLL-13-T gram-negative coccus has the following characteristics:
1) morphological characteristics:
gram-negative cocci, the colony on the solid culture medium is light pink, round and convex, the edge is neat, and the surface is moist and sticky; but the colony color is lighter than that of the original strain, the surface viscosity is lower than that of the original strain, and the cell morphology is relatively large;
2) physiological and biochemical characteristics
The mutant strain is microaerophilic strain, and has positive catalase, oxidase, phosphatase, alcohol dehydrogenase and urease, and cannot reduce nitrate and produce H2S, the growth rate of the mutant strain is higher than that of the growing strain;
3) carbon source utilization
Taking glucose, sucrose, arabinose, lactose, D-galactose and D-maltose as carbon sources; carbon sources such as L-rhamnose, D-mannose, D-xylose, melibiose, citric acid, lysine, ornithine and arginine cannot be utilized.
In a third aspect, the invention provides a process for producing coenzyme Q10 of a rhodobacter sphaeroides mutant strain according to the second aspect, comprising the steps of:
1) marking the rhodobacter sphaeroides mutant strain on a solid medium flat plate, and standing and culturing at 32 ℃;
2) first-order seed culture: picking the activated single colony on the plate, transferring the single colony to a 250mL conical flask containing 50mL of seed culture medium, and culturing for 24h under the conditions of 32 ℃ and 200 r/min;
3) secondary seed culture: transferring all the primary seed liquid into a 1L conical flask containing 450mL of seed culture medium, and continuously culturing for 20-22h at 32 ℃ under the condition of 200 r/min;
4) fermentation culture: transferring the secondary seed liquid into a 10L fermentation tank containing a fermentation culture medium, wherein the liquid loading amount is 4.5L, the fermentation temperature is 32 ℃, the initial stirring speed is 500r/min, and the aeration ratio is 2.0 vvm; after 48 hours, adjusting the stirring speed to 400r/min, the aeration ratio to 0.75vvm and the fermentation time to 120 hours;
5) the cells were harvested by centrifugation, and the coenzyme Q10 was extracted.
Further, 1) in the culture medium, the culture medium is subjected to static culture at 32 ℃ for 4 to 5 days.
Further, ammonia water is adopted in the step 4) to regulate the pH value to be about 7.2.
Further, in 4), an exponential feed supplement mode is adopted to maintain the glucose at 10 g/L.
Further, the solid medium includes: 3g/L glucose, 8g/L yeast powder, 2g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L, biotin 15 mu g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, agar powder 15g/L, and pH 7.2; the seed culture medium comprises: 3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, 2.0g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L，FeSO4100mg/L，MnSO41.0mg/L, biotin 15 mug/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2; the fermentation medium comprises: 40g/L glucose, 5.5g/L corn steep liquor dry powder, 3.0g/L sodium glutamate, 2g/L NaCl, CaCl20.1g/L，KH2PO42.5g/L,MgSO46.3g/L，NH4SO42.5g/L，FeSO4100mg/L，MnSO41mg/L，CoCl220mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
Further, coenzyme Q10 is extracted by an ultrasonic-assisted method in the step 5).
As a preferred embodiment, the extraction of coenzyme Q10 by using the ultrasonic-assisted method comprises the steps of taking a zymocyte suspension, adding appropriate 20mmol/L HCl, fully mixing, carrying out water bath at 75 ℃ for 15min, carrying out centrifugation at 5000r/min for 10min, discarding the supernatant, adding appropriate extracting solution (ethyl acetate: ethanol is 5:3, v/v), fully mixing, then placing in an ultrasonic cleaner, carrying out ultrasonic-assisted extraction for 10min, and carrying out light-shielding extraction for 15 min. Centrifuging the extractive solution at 8000r/min for 15min to obtain coenzyme Q10 extractive solution. It is to be understood by those skilled in the art that the present invention can extract coenzyme Q10 by any method, and is not limited to the method of the present application, as long as coenzyme Q10 can be extracted.
In a fourth aspect, the invention provides the use of a mutant rhodobacter sphaeroides strain according to the second aspect of the invention for the production of coenzyme Q10.
The invention has the advantages and beneficial effects that:
the invention is mutagenized by a chemical mutagen, and screened by taking coenzyme Q10 structural analogues or precursor analogues as screening markers, and a mutant strain with low carotenoid synthesis, low viscosity, high growth rate and high coenzyme Q10 accumulation is found for the first time, is named as Rhodobacter sphaeroides (Rhodobacter sphaeroides) YLL-13-T, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number of CGMCC No. 18983. Compared with the original strain, the biomass of the mutant strain, the yield of the coenzyme Q10 and the yield of the coenzyme Q10 are respectively increased by 12.3 percent, 42.5 percent and 23.6 percent compared with the original strain.
The invention optimizes the production process of coenzyme Q10, and the fermentation unit of coenzyme Q10 is improved by 2.1 times by utilizing the optimized culture medium and the optimized production process.
Biological material preservation instructions
Name: rhodococcus sphaeroides (Rhodobacter sphaeroides) YLL-13-T;
the preservation unit: china general microbiological culture Collection center
The preservation unit is abbreviated as: CGMCC;
the preservation number is: CGMCC No. 18983;
the preservation date is as follows: 11 and 20 months in 2019
Drawings
FIG. 1 is an electron scanning electron micrograph of somatic cells of R.sphaeroides YLL-13 and YLL-13-T, in which FIG. a shows YLL-13 and FIG. b shows YLL-13-T.
FIG. 2 is an HPLC chart of coenzyme Q10.
FIG. 3 is an HPLC-MS spectrum of coenzyme Q10 in rhodobacter sphaeroides cells.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
EXAMPLE 1 screening of Rhodobacter sphaeroides mutant strains
A Rhodobacter sphaeroides mutant strain is screened by taking a Rhodobacter sphaeroides original strain (Rhodobacter sphaeroides YLL-13) separated from sludge of a sewage treatment plant as an initial strain, and the steps are as follows:
1. preparation of the culture Medium
1) Solid medium: 3g/L glucose, 8g/L yeast powder, 2g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L, biotin 15 mu g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, agar powder 15g/L, and pH 7.2.
2) Seed culture medium: 3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, 2.0g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L，FeSO4100mg/L，MnSO41.0mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
2. The original strain YLL-13 is inoculated in a seed culture medium and cultured for 24 hours at the temperature of 32 ℃ and under the condition of 200 r/min.
3. Taking 1mL of bacterial suspension, centrifuging at 5000r/min for 5min to obtain thalli, centrifuging and washing twice with physiological saline, then suspending in physiological saline containing 0.6mg/mL of nitrosoguanidine, and standing at room temperature for 45 min.
4. Centrifuging at 5000r/min for 5min to obtain thallus, resuspending with physiological saline, spreading on solid plate containing 12mg/L vitamin K3 and 0.6g/L p-hydroxybenzoic acid, and standing at 32 deg.C for 4-5 days.
5. Selecting mutant strains with large colonies, relatively light colors and relatively low colony viscosity on the plate, culturing for 120h at 32 ℃ under 200r/min in a seed culture medium, and determining the content of the cell coenzyme Q10.
6. Mutant strains with higher coenzyme Q10 yield are screened, passaged for 10 times, and the coenzyme Q10 content, colony morphology and growth rate change are monitored.
7. Screening and preserving strains with good genetic stability, low carotenoid color yield, low viscosity, high growth rate and high coenzyme Q10 yield, wherein the strains are named as: YLL-13-T; the preservation unit is China general microbiological culture Collection center, and the preservation numbers are: CGMCC No. 18983; the preservation date is as follows: 11/20/2019.
8. And detecting the thallus morphology of thallus cells after the starting strain and the mutant strain are cultured in a seed culture medium for 24 hours by using an electronic scanning electron microscope.
The YLL-13-T strain of the invention has the following properties:
YLL-13-T is gram-negative coccus, the bacterial colony on the solid culture medium is light pink, is round and convex, has neat edges, and has moist and viscous surface; but the colony color is lighter than that of the original strain, the surface viscosity is lower than that of the original strain, the growth rate of the mutant strain is higher than that of the original strain, and the cell morphology is relatively large (figure 1); the mutant strain is microaerophilic strain, and has positive catalase, oxidase, phosphatase, alcohol dehydrogenase and urease, and cannot reduce nitrate and produce H2S。
Example 2 fermentation of YLL-13 to produce coenzyme Q10
1. Fermentation culture
Selecting activated YLL-13 colony which is subjected to static culture for 4-5 days at 32 ℃ on a solid plate, inoculating the colony in a 250mL conical flask containing 50mL of seed culture medium, and culturing for 24h at 32 ℃ under the condition of 200 r/min; then, 50mL of the bacterial suspension is completely transferred to a 1L conical flask containing 450mL of seed culture medium, and the culture is continued for 120h under the conditions of 32 ℃ and 200 r/min.
The seed culture medium comprises the following components: 3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, 2.0g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L，FeSO4100mg/L，MnSO41.0mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
2. Extraction of coenzyme Q10
Adding 200 mu L of 20mmol/L HCl into 1mL of zymocyte suspension, fully mixing, carrying out water bath at 75 ℃ for 15min, centrifuging at 5000r/min for 10min, discarding the supernatant, adding 5mL of extracting solution (ethyl acetate: ethanol is 5:3, v/v), fully mixing, placing in an ultrasonic cleaner, carrying out ultrasonic-assisted extraction for 10min, and carrying out light-shielding extraction for 15 min. Centrifuging the extractive solution at 8000r/min for 15min to obtain coenzyme Q10 extractive solution.
3. Characterization of coenzyme Q10
Qualitative analysis of coenzyme Q10 was performed by liquid chromatography-mass spectrometry (HPLC-MS).
The chromatographic conditions comprise that a Hypersil ODS (C18) chromatographic column (4.6mm × 150mm,5 mu m), pure methanol as a mobile phase, the column temperature of 40 ℃, the detection wavelength of 275nm, the flow rate of 1mL/min, the sample injection amount of 10 mu L, an electrospray ion source (ESI) as an ion source, detection by adopting a positive ion mode, the scanning range of 100-1800m/z, the drying gas flow of 6L/min, the atomization gas pressure of 20psi and the capillary voltage of 4000V.
4. Quantification of coenzyme Q10
Coenzyme Q10 was quantitatively analyzed by High Performance Liquid Chromatography (HPLC).
Chromatographic conditions are Hypersil ODS (C18) chromatographic column (4.6mm × 150mm,5 μm), mobile phase methanol/isopropanol (3: 1 (v/v)), column temperature 40 ℃, detection wavelength 275nm, flow rate 1mL/min, and sample injection amount 20 μ L.
5. Results
The quantitative and qualitative results are shown in FIGS. 2 and 3, respectively, and the fermentation unit of Rhodobacter sphaeroides YLL-13 coenzyme Q10 is 0.23 g/L.
Example 3 optimized Process for fermentation of coenzyme Q10 from YLL-13
1. Preparation of the culture Medium
1) Seed culture medium
3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, 2.0g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L，FeSO4100mg/L，MnSO41.0mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, sulfur hydrochlorideAmine 1mg/L, pH7.2.
2) Fermentation medium
40g/L glucose, 5.5g/L corn steep liquor dry powder, 3.0g/L sodium glutamate, 2.0g/L NaCl, CaCl20.1g/L，KH2PO42.5g/L,MgSO46.3g/L，NH4SO42.5g/L，FeSO4100mg/L，MnSO41mg/L，CoCl220mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
2. Fermentation culture
1) First class seed
Activated YLL-13 colonies cultured on solid plates at 32 ℃ for 4-5 days were picked, inoculated into 250mL Erlenmeyer flasks containing 50mL seed medium, and cultured at 32 ℃ at 200r/min for 24 hours.
2) Second grade seed
Transferring 50mL of the bacterial suspension of the first-order seeds into a 1L conical flask containing 450mL of seed culture medium, and continuously culturing for 20-22h under the conditions of 32 ℃ and 200 r/min.
3) Fermentation culture
Transferring all the 500mL secondary seed liquid into a 10L fermentation tank, wherein the liquid loading amount is 4.5L, the fermentation temperature is 32 ℃, the aeration ratio is 2.0vvm, and the initial stirring speed is 500 r/min; after 48h, the stirring rate was adjusted to 400r/min and the aeration ratio was 0.75 vvm. The pH value is regulated to be about 7.2 by ammonia water. When the glucose content of the fermentation system is lower than 10g/L, an exponential flow feeding manner is adopted to maintain the glucose at 10 g/L. The fermentation time is 120 h. And after the fermentation is finished, centrifuging to obtain thalli, and extracting with the assistance of ultrasonic to obtain the coenzyme Q10.
3. Extraction of coenzyme Q10
Adding 200 mu L of 20mmol/L HCl into 1mL of zymocyte suspension, fully mixing, carrying out water bath at 75 ℃ for 15min, centrifuging at 5000r/min for 10min, discarding the supernatant, adding 5mL of extracting solution (ethyl acetate: ethanol is 5:3, v/v), fully mixing, placing in an ultrasonic cleaner, carrying out ultrasonic-assisted extraction for 10min, and carrying out light-shielding extraction for 15 min. Centrifuging the extractive solution at 8000r/min for 15min to obtain coenzyme Q10 extractive solution.
4. Quantification of coenzyme Q10
Coenzyme Q10 was quantitatively analyzed by High Performance Liquid Chromatography (HPLC).
Chromatographic conditions are Hypersil ODS (C18) chromatographic column (4.6mm × 150mm,5 μm), mobile phase methanol/isopropanol (3: 1 (v/v)), column temperature 40 ℃, detection wavelength 275nm, flow rate 1mL/min, and sample injection amount 20 μ L.
5. Results
Under the fermentation condition, the fermentation unit of rhodobacter sphaeroides YLL-13 coenzyme Q10 reaches 0.73g/L, the biomass is 72.4g/L, the yield of coenzyme Q10 is 10.08mg/g (DCW), and the yield is far greater than that of unoptimized YLL-13 coenzyme Q10.
Example 4 optimized Process for production of coenzyme Q10 by fermentation of YLL-13-T
1. Preparation of the culture Medium
1) Seed culture medium
3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, 2.0g/L NaCl, KH2PO41.3g/L，MgSO4125mg/L，FeSO4100mg/L，MnSO41.0mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
2) Fermentation medium
40g/L glucose, 5.5g/L corn steep liquor dry powder, 3.0g/L sodium glutamate, 2.0g/L NaCl, CaCl20.1g/L，KH2PO42.5g/L,MgSO46.3g/L，NH4SO42.5g/L，FeSO4100mg/L，MnSO41mg/L，CoCl220mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
2. Fermentation culture
1) First class seed
Activated YLL-13-T colonies cultured on solid plates at 32 ℃ for 4-5 days are picked, inoculated into 250mL Erlenmeyer flasks containing 50mL seed medium, and cultured at 32 ℃ and 200r/min for 24h
2) Second grade seed
Transferring 50mL of the bacterial suspension of the first-order seeds into a 1L conical flask containing 450mL of seed culture medium, and continuously culturing for 20-22h under the conditions of 32 ℃ and 200 r/min.
3) Fermentation culture
Transferring all the 500mL secondary seed liquid into a 10L fermentation tank, wherein the liquid loading amount is 4.5L, the fermentation temperature is 32 ℃, the aeration ratio is 2.0vvm, and the initial stirring speed is 500 r/min; after 48h, the stirring rate was adjusted to 400r/min and the aeration ratio was 0.75 vvm. The pH value is regulated to be about 7.2 by ammonia water. When the glucose content of the fermentation system is lower than 10g/L, an exponential flow feeding manner is adopted to maintain the glucose at 10 g/L. The fermentation time is 120 h. And after the fermentation is finished, centrifuging to obtain thalli, and extracting with the assistance of ultrasonic to obtain the coenzyme Q10.
3. Extraction of coenzyme Q10
Adding 200 mu L of 20mmol/L HCl into 1mL of zymocyte suspension, fully mixing, carrying out water bath at 75 ℃ for 15min, centrifuging at 5000r/min for 10min, discarding the supernatant, adding 5mL of extracting solution (ethyl acetate: ethanol is 5:3, v/v), fully mixing, placing in an ultrasonic cleaner, carrying out ultrasonic-assisted extraction for 10min, and carrying out light-shielding extraction for 15 min. Centrifuging the extractive solution at 8000r/min for 15min to obtain coenzyme Q10 extractive solution.
4. Quantification of coenzyme Q10
Coenzyme Q10 was quantitatively analyzed by High Performance Liquid Chromatography (HPLC).
Chromatographic conditions are Hypersil ODS (C18) chromatographic column (4.6mm × 150mm,5 μm), mobile phase methanol/isopropanol (3: 1 (v/v)), column temperature 40 ℃, detection wavelength 275nm, flow rate 1mL/min, and sample injection amount 20 μ L.
5. Results
Under the fermentation condition, the biomass of the mutant strain, the yield of coenzyme Q10 and the yield of coenzyme Q10 after the rhodobacter sphaeroides YLL-13-T coenzyme Q10 is fermented for 120 hours are 83.5g/L, 1.04g/L and 12.46mg/g (DCW), respectively; compared with the original strain, the strain is respectively improved by 12.3 percent, 42.5 percent and 23.6 percent.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> university of eight agricultural reclamation of Heilongjiang
<120> rhodobacter sphaeroides mutant strain for producing coenzyme Q10 and method for producing coenzyme Q10 through fermentation
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1428
<212>DNA
<213>Rhodobacter sphaeroides
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agagtttgat cctggctcag aatgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
gaagtcttcg gacttagcgg cggacgggtg agtaacgcgt gggaacgtgc cctttgcttc 120
ggaatagccc cgggaaactg ggagtaatac cgaatgtgcc ctttggggga aagatttatc 180
ggcaaaggat cggcccgcgt tggattaggt agttggtggg gtaatggcct accaagccga 240
cgatccatag ctggtttgag aggatgatca gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagtg gggaatctta gacaatgggc gcaagcctga tctagccatg 360
ccgcgtgatc gatgaaggcc ttagggttgt aaagatcttt caggtgggaa gataatgacg 420
gtaccaccag aagaagcccc ggctaactcc gtgccagcag ccgcggtaat acggaggggg 480
ctagcgttat tcggaattac tgggcgtaaa gcgcacgtag gcggatcgga aagtcagagg 540
tgaaatccca gggctcaacc ctggaactgc ctttgaaact cccgatcttg aggtcgagag 600
aggtgagtgg aattccgagt gtagaggtga aattcgtaga tattcggagg aacaccagtg 660
gcgaaggcgg ctcactggct cgatactgac gctgaggtgc gaaagcgtgg ggagcaaaca 720
ggattagata ccctggtagt ccacgccgta aacgatgaat gccagtcgtc gggcagcatg 780
ctgttcggtg acacacctaa cggattaagc attccgcctg gggagtacgg ccgcaaggtt 840
aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgca gaaccttacc aacccttgac atggcgatcg cggttccaga gatggttcct 960
tcagttcggc tggatcgcac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttcggttaa gtccggcaac gagcgcaacc cacgtcctta gttgccagca ttcagttggg 1080
cactctaggg aaactgccgg tgataagccg gaggaaggtg tggatgacgt caagtcctca 1140
tggcccttac gggttgggct acacacgtgc tacaatggca gtgacaatgg gttaatccca 1200
aaaagctgtc tcagttcgga ttggggtctg caactcgacc ccatgaagtc ggaatcgcta 1260
gtaatcgcgt aacagcatga cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320
cacaccatgg gaattggttc tacccgaagg cggtgcgcca acctcgcaag aggaggcagc 1380
cgaccacggt aggatcagtg actggggtga agtcgtaaca aggtaacc 1428
Claims (6)
1. A Rhodobacter sphaeroides mutant strain is characterized in that the Rhodobacter sphaeroides mutant strain is named Rhodobacter sphaeroides YLL-13-T, is preserved in China general microbiological culture Collection center, and has a preservation number of CGMCC No. 18983.
2. The process for producing coenzyme Q10 of a rhodobacter sphaeroides mutant strain according to claim 1, comprising the steps of:
1) marking the rhodobacter sphaeroides mutant strain on a solid medium flat plate, and standing and culturing at 32 ℃;
2) first-order seed culture: picking the activated single colony on the plate, transferring the single colony to a 250mL conical flask containing 50mL of seed culture medium, and culturing for 24h under the conditions of 32 ℃ and 200 r/min;
3) secondary seed culture: transferring all the primary seed liquid into a 1L conical flask containing 450mL of seed culture medium, and continuously culturing for 20-22h at 32 ℃ under the condition of 200 r/min;
4) fermentation culture: transferring the secondary seed liquid into a 10L fermentation tank containing a fermentation culture medium, wherein the liquid loading amount is 4.5L, the fermentation temperature is 32 ℃, the initial stirring speed is 500r/min, and the aeration ratio is 2.0 vvm; after 48 hours, adjusting the stirring speed to 400r/min, the aeration ratio to 0.75vvm and the fermentation time to 120 hours;
5) the cells were harvested by centrifugation, and the coenzyme Q10 was extracted.
3. The process of claim 2, wherein the glucose level in 4) is maintained at 10g/L by feeding the glucose in an exponential manner.
4. The process according to claim 3, wherein the pH is adjusted to 7.2 with ammonia.
5. The production process according to any one of claims 2 to 4, wherein the solid medium comprises: 3g/L glucose, 8g/L yeast powder, 2g/L NaCl, KH2PO41.3 g/L，MgSO4125mg/L, biotin 15 mu g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, agar powder 15g/L, and pH 7.2; the seed culture medium comprises: 3g/L glucose, 8g/L yeast powder, 1.0g/L corn steep liquor dry powder, 2.0g/L NaCl, KH2PO41.3 g/L，MgSO4125 mg/L，FeSO4100 mg/L，MnSO41.0mg/L, biotin 15 mug/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2; the fermentation medium comprises: 40g/L glucose, 5.5g/L corn steep liquor dry powder, 3.0g/L sodium glutamate, 2g/L NaCl, CaCl20.1 g/L，KH2PO42.5g/L, MgSO46.3 g/L，NH4SO42.5 g/L，FeSO4100 mg/L，MnSO41 mg/L，CoCl220mg/L, biotin 15. mu.g/L, nicotinic acid 1mg/L, thiamine hydrochloride 1mg/L, pH 7.2.
6. Use of a mutant rhodobacter sphaeroides strain according to claim 1 for the production of coenzyme Q10.
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